Temporins, antimicrobial peptides from the European red

Result
The antibacterial activity of temporins A and B was also tested against three cell-wall defective mutant strains of E. coli D21, D21e7, D21f1, and D21f2, which have lost increasing amounts of sugar residues of their lipopolysaccharide chain. Medium E was found to increase the activity of temporins in all strains tested. However,no similar effects were observed with gram-positive bacteria.
Result
first tested against B. meguteriurn and E.coli D21: Temporins A, B, F, G, and L were active on both bacterial strains, but temporins C, D, E, H, and K only showed some activity against B. meguleriurn, the most sensitive bacterium. The antimicrobial activity of synthetic temporins A and B, expressed as lethal concentration values, is reported in Table 2
Disscussion
Most of the temporins have antibacterial activity, in particular those that contain a positively charged amino acid and thus a net charge of +2. When the basic residue is replaced by an asparagine, or, when three residues inside the molecule are deleted, the molecule becomes inactive. Temporins are the shortest natural antibacterial peptides that have been found to date. Five 13 - 15-residue cecropin-melittin hybrids were found to be almost equally active as the longer parental molecules. It seems that the minimal requirement for antibacterial activity is 13 residues and a net basic charge is also needed.
Northern blot analysis
Poly(A)-rich RNA (5 μg) was fractionated by electrophoresis Blotted directly onto Nytran sheets Probe: The insert of clone Rt-17 labelled by random priming. Hybridization
Temporins, antimicrobial peptides from the European red frog Rana temporaria
05基地班 魏潇 冯秀云 程超 靳利蕊 张蓉 卢会文
Introduction
Skin secretions of frogs contain many different types of antibacterial peptides. In particular, a variety of such peptides has been isolated from several Rana species. There are 4 different groups: brevinin 1 Family, brevinin 2 Family, 37-residue peptides, a group that consists of esculentin 1--has the highest antibacterial activity. The signal sequences of precursors of antimicrobial peptides from Rana species were very similar to those with different biological activities from Phyllornedusa species. Search for the precursors of new peptides in the skin of Rana temporuria, whose signal peptide is similar to signal peptide of esculentin 1.
Antimicrobial assay
The antibacterial activity was tested against Bacillus megaterium BM11, Staphylococcus aureus Cowan1, Yersinia pseudotubercolosis, Streptococcus pyogenes hemolytic group A, Pseudonzonas aeruginosa ATCC 15692, Escherichia coli D21, E. coli D21e7, E. coli D21f1, E. coli D21f2 and E. coli D22, using an inhibition zone assay.
Build up a cDNA library of R.t
Find a positive clone with a insert which encode a new peptide
Find the natural peptide from the skin secretion of R.t Analyse the structure of the new peptide and test the antibactieral activity of the newrobe derived from clone Rt-17 recognized an abundant message, detected as a single, rather broad band in the range 400-500 nucleotides. Under the same conditions, no signal could be obtained from the skin of other amphibian species.
Methods And Result
Isolation of RNA and cDNA screen Northern blot analysis Collection and purification of skin secretions and Structural analysis Antimicrobial assay
Isolation of RNA and cDNA screen
Isolation of poly(A)-rich RNA by affinity chromatography Preparation of the cDNA library Preparation of probe: a 240-bp fragment obtained by digestion of the esculentin 1 cDNA with HindIII, then labelled by random priming. Screening Positive clones were selected and analysed by cleavage with restriction enzymes and nucleotide sequencing.
Result
Six positive clones were detected. The sequences of the inserts present in clones Rt-5, Rt-6, and Rt17 are shown in Fig. 1. The predicted end products would be amidated peptides containing 13 amino acids for clones Rt-5 and Rt-17, while Rt-6 has a 9 bp deletion in this region, and thus codes only for a decapeptide.
Result
20 mg lyophilized material could be obtained; Preliminary HPLC purification; Each fraction was subjected to N-terminal analysis, to identify those with N-terminal leu or phe as predicted from the cDNA sequences; Further purified by HPLC and subjected to amino acid and sequence analysis; 3 predicted peptides were found to be present in the secretion; Structurally related to these peptides, were also isolated and termed temporins.